The role of GTP in choleragen-dependent activation of adenylate cyclase was studied by examining the effect of nucleoside triphosphates on choleragen-catalyzed ADP-ribosylation of proteins. Choleragen catalyzed the transfer of ADP-ribose from NAD to multiple soluble and membrane proteins of bovine thymus and brain as well as to purified proteins such as polyariginine, lysozyme, and histone. Nucleoside triphosphates, primarily GTP, enhanced the labeling of several soluble proteins, and had variable effects on ADP-ribosylation of purified proteins.